The depressed Ca-cycling in animal models of heart failure and human failing hearts has been suggested to reflect, at least in part, the impaired Ca-cycling by the sarcoplasmic reticulum (SR). There are three major functions of the SR: a) Ca-uptake from the cytosol into the SR lumen resulting in muscle relaxation; b) Ca-storage in the SR lumen; and c) Ca-release from the SR into the cytosol resulting in muscle contraction. The main SR proteins responsible for these functions are: the Ca-transport ATPase (SERCA2) with its regulator phospholamban (PLN); the Ca-storage proteins, calsequestrin and a histidine rich Ca-binding protein (HRC); and the Ca-release ensemble composed of the ryanodine receptor, junctin and triadin. In this project, we propose further studies on elucidating the regulatory role of increased (chronic or acute) PLN phosphorylation, through regulation of its phosphatase 1 activity by Inhibitor-I, in the control of contractility and the heart's responses to stress. Furthermore, since alterations in the degree of PLN phosphorylation reflect alterations in SR Ca-load and release, we propose to elucidate the functional roles of: a) SR Ca load through calsequestrin, the major Ca storage protein in the SR lumen; and b) SR Ca-release through junctin, one of the major proteins in the release cassette. Animal models with reduced or ablated expression of calsequestrin or junctin will be generated and their cardiac phenotypes will be analyzed at the molecular, subcellular, cellular, organ and intact animal levels under basal and stress conditions. These studies will provide important information on the functional role of calsequestrin and junctin in vivo under physiological and pathophysiological conditions. Overall, our proposed studies will advance our knowledge on the mechanisms underlying regulation of Ca homeostasis by the SR function in the mammalian heart. They will also provide valuable insights into the crosstalk between the various SR Ca handling proteins and their regulatory effects on cardiac contractility. [unreadable] [unreadable]